In yeast genetic systems, many options are available for delivery of gene sequences for the purpose of conferring a phenotype onto the host cell. For example, one common delivery system is a high copy plasmid system based on the endogenous yeast 2-micron plasmid. Plasmids from this origin achieve copy numbers of roughly 100 per cell and are randomly segregated to daughter cells upon division. In another system, the CEN system, CEN plasmids are maintained at low copy number (approximately 1 to 2 per cell) are segregated to daughter cells by the same mechanism used for segregation of the host chromosomes.
Further, methods have been devised in yeast by which the problems of gene isolation and discovery of gene function can be addressed efficiently. For example, in yeast it is possible to isolate genes via their ability to complement specific phenotypes. Further, in yeast, targeted insertional mutagenesis techniques can be used in yeast to inactivate or "knock out" a gene's activity. In mammalian systems, however, such methods are, in practical terms, lacking, which has made the elucidation of mammalian gene function a very difficult task.
For example, with respect to gene inactivation techniques in mammalian cells, the fact that mammalian cells are diploid and have complex genomes cause insertional mutagenesis techniques in mammalian systems to be a laborious, time-consuming and lengthy process.
Further, a major barrier to the development of such capabilities as complementation screening in mammalian cells has been that conventional techniques yield gene transfer efficiencies in most cells (0.01%-0.1%) that make screening of high complexity libraries impractical. While reports indicate that recombinant, replication deficient retroviruses can make possible increased gene transfer efficiencies in mammalian cells (Rayner & Gonda, 1994, Mol. Cell. Biol. 14:880-887; Whitehead et al., 1995, Mol. Cell. Biol. 15:704-710), retroviral-based functional mammalian cloning systems are inconvenient and have, thus far, failed to achieve widespread use.
The lack of convenience and impracticality of current retroviral-based cloning systems include, for example, the fact that the production of high complexity libraries has been limited by the low transfection efficiency of known retroviral packaging cell lines. Furthermore, no system has provided for routine, easy recovery of integrated retroviral proviruses from the genomes of positive clones. For example, in currently used systems the recovery of retrovirus inserts may be accomplished by polymerase chain reaction (PCR) techniques, however this is quite time consuming and variable for different inserts. Furthermore, with the use of PCR, additional cloning steps are still required to generate viral vectors for subsequent screening. Additionally, no mechanism has been available for distinguishing revertants from provirus-dependent rescues, a major source of false positives.
Further, it would be advantageous if an episomal system such as those found in yeast existed for efficient, broad spectrum use in mammalian systems. While bovine papillomaviruses (BPV), for example, replicate as extrachromosomal episomes, their use in developing episomal vectors has been limited.
Specifically, the ability of BPV replicate as episomes has been exploited in the past to create episomal vectors, using the so-called 69% fragment (T69). Vectors based upon T69 replicate in certain murine cell lines to give copy numbers that range from 15 to 500 copies per haploid genome, depending on the cell line. T69 vectors, however, exhibit a narrow host range. Further, the T69 fragment, like SV40, is oncogenic. Indeed, one method for identifying cells carrying T69 vectors specifically involves screening for transformed C127 cells.